Therapeutic use of aerosolized s-nitrosoglutathione in cystic fibrosis

ABSTRACT

Composition comprising nitrosylating agent is administered to a patient having cystic fibrosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/240,708, filed Oct. 16, 2000 which is incorporatedherein by reference.

BACKGROUND

[0002] Cystic fibrosis (CF) is an autosome recessive disease. Affectedpatients have an average life expectancy of 30 years. This earlymortality is primarily related to lung disease, in particular for 95% ofpatients, death results from progressive respiratory failure associatedwith impaired mucus clearance and excessive overgrowth of bacteria andfungi in the airways. The lung disease is characterized by 1) abnormalsalt and water transport in the airway epithelium, such that too muchsodium and too little chloride crosses the epithelial membrane; 2) adefect in glutathione transport out of airway epithelial cells; 3)inspissated secretions and oxidant damage, in part related to salt,water, and glutathione transport abnormalities; 4) impaired ciliarymotility associated with thick secretions; 5) chronic colonization withdenitrifying organisms such as Pseudomonas aeruginosa and Aspergillusfumigatus; and 6) chronic bronchoconstriction associated withrecruitment of neutrophils and other inflammatory cells and associatedrelease of bronchoconstricting mediators and, as a result of all ofthese factors, chronic, progressively worsening, dyspnea.

[0003] Cystic fibrosis is caused by mutations in a membrane-associatedchloride channel, the cystic fibrosis transmembrane regulatory protein(CFTR). The most common mutation, accounting for 70% of alleles, is a 3base pair (single amino acid) deletion at the 508 position on the firstATP binding domain, the (ΔF508) mutation. This is referred to as a classII mutation in that intact and potentially functional protein isdegraded in the endoplasm reticulum because of a small folding error. Itis known that his degradation can be bypassed in vitro by hypothermiaand by treatment with compounds such as glycerol and phenylbutyrate.These treatments result in functional expression of (ΔF508) CFTR on thecell surface. It has recently been proposed that CFTR degradation in theendoplasmic reticulum (ER) is mediated by the ubiquitin-proteasomesystem. We have preliminary evidence that S-nitrosoglutathione (GSNO)inhibits the ubiquitin proteosome pathway, stabilizing the expression ofpost-translationally/degradation-regulated proteins such as hypoxyinducible factor 1-μ.

[0004] Furthermore, GSNO is of interest in cystic fibrosis for severalestablished reasons. We have shown in 1993, that it is a compoundpresent endogenously in the airways and has activity as abronchodilator. GSNO is also known to increase ciliary beat frequency,augmenting airway clearance to inhibit amiloride-sensitive sodiumtransport, augmenting airway lining fluid salt and water retention. Ithas established antimicrobial properties, inhibiting the replication ofviruses, bacteria, and parasites. We have recently discovered thatlevels of GSNO are low in the bronchoalveolar lavage fluid of patientswith mild cystic fibrosis. All of these established functions could havea salutary effect in CF.

[0005] Surprisingly, we have have discovered that the treatment of cellshomozygous to the (ΔF508) mutation with 100 μM GSNO increases theexpression of CFTR and increases the maturation of CFTR (see FIG. 1). Wehave also discovered that inhalation of GSNO at concentrations effectiveto enhance the production of CFTR (a dose of about 0.05 ml/kg of a 10 mMGSNO solution) results in improved oxygenation. Finally we have shownthat GSNO is well tolerated in CF and has no adverse systemic affects.Therefore, since replacement of low levels of GSNO is well tolerated,acutely beneficial, and can increase the amount of functional CFTRexpressed on the cell surface, its use is proposed to benefit peoplewith the disease.

BRIEF SUMMARY OF INVENTION

[0006] The present invention is directed to composition and method fortreating CF patients. The method comprises the delivery ofS-nitrosoglutathione (GSNO) in concentrations equal to or in excess of500 nmole/kg (175 mcg/kg), or other nitrosylating agents such as ethylnitrite, to epithelial surfaces of patients with cystic fibrosis for thepurpose of insuring adequate concentrations of S-nitrosylating agent onthe epithelial surfaces of CFTR and epithelial function. Thecompositions of the present invention comprise a nitrosonium donorincluding, but not limited to GSNO and other S-nitrosothiols (SNOs) in apharmaceutically acceptable carrier that allows for administration bynebulized or other aerosol treatment to patients with cystic fibrosis.

BRIEF SUMMARY OF THE DRAWINGS

[0007]FIG. 1. S-Nitrosoglutathione stabilizes and allows maturation ofhuman CFTR protein beating the ΔF508 mutation. Human pancreaticadenocarcinoma cells from a patient homozygous for ΔF 508 (CFPAC-1) weregrown to confluence and treated with medium alone (control) or 500 nM, 1μM, 5 μM, 10 μM or 100 mM GSNO for 6 hours. The cells were harvested,lysed, resuspended in NP40 buffer and underwent Western Blot analysiswith anti-CFTR antibody. Results are shown in FIG. 1 where Lane A showsexpression of the immature non-glycosylated protein (140 kDa), Lane Bshows expression of partially matured protein (about 160 kDa), and LaneC shows expression of mature protein (180 kDa).

[0008] Increased expression of the immature non-glycosylated protein(140 kDa) reflects decreased degradation of the immature protein in theendoplasma retimulum. The appearance of a band at 180 kDa (matureprotein) reflects maturation of the protein in the endoplasmic reticuluman expression of a form capable of being expressed on the cell surfacerepresentative of three experiments. The fill maturing of the proteinaccommodates for the ΔF 508 mutation.

[0009]FIG. 2. S-Nitrosothiol levels are low in the cystic fibrosis anway. Subjects with mild cystic fibrosis and control subjects underwentbronchoalveolar lavage. Lavage fluid was frozen (−80° C.) andsubsequently underwent blinded analysis for S-nitrosothiol content usingan established method of reduction (1 mM cysteine, 100 μM CuCl; 50° C.,pH 6.5) to nitric oxide followed by chemiluminescent analysis.S-Nitrosothiols were only measurable in two CF patients, and Rank Sumanalysis showed that values were lower than in controls (p<0.01).

[0010]FIG. 3. Inhalation of 0.05 ml/kg of 10 mM GSNO results in improvedoxygenation in patients with CF. In a double-blind, placebo-controlledstudy, patients with CF received GSNO in 10 mM phosphate buffered saline(PBS) (n=9) or 10 mM PBS alone (n=11) by nebulizer. The two groups werematched with respect to age, vital signs, oxygen saturation, vitalcapacity, FEV₁, and FEF₂₅₋₇₅ (p=7). Change in oxygen saturation frompretreatment baseline is recorded as a function of time. Results areshown in FIG. 3 where the continuous line represents the GSNO group andthe line composed of dashes represents the PBS alone group. As shown inFIG. 3, subjects in the GSNO group had a greater improvement in oxygensaturation than those in the PBS group that was sustained throughout thestudy (p<0.001 by ANOVA).

[0011]FIG. 4. Inhaled S-nitrosoglutathione cases increased expirednitric oxide (NO) concentration in CF. Data presented represent the twogroups described in FIG. 3, but with respect to expired nitric oxideconcentration (measured off-line with chemiluminescence in accordancewith American Thoracic Society guidelines). Results are shown in FIG. 4where the continuous line represents the GSNO group and the linecomposed of dashes represents the PBS alone group. As shown in FIG. 4,change in expired NO from baseline is higher in the GSNO group at alltime points (p<0.001 by ANOVA). Note that the change at 30 minutes isless pronounced than at 5 minutes, though improvement in oxygenation isas dramatic at 30 minutes as at 5 minutes. This finding, coupled withthe relatively low levels of expired NO after GSNO treatment, suggeststhat the effect of GSNO on improved oxygenation does not require itsbreakdown to form the nitric oxide radical but may, instead, involvetransnitrosation reactions with airway proteins. These S-nitrosylationreactions may be critically important to smooth muscle relaxation,antimicrobial effects and modification of ion channel function.

[0012]FIGS. 5A, 5B, 5C, 5D, 5E and 5F. Inhaled S-nitrosoglutathione iswell tolerated in CF patients. As shown in FIGS. 5A, 5B, 5C, 5D, 5E, and5F, where the continuous lines represent the GSNO group and the linescomposed of dashes represent the PBS alone group, no acute change invital signs or lung function was observed in patients receiving GSNOusing the study protocol described in conjunction with FIG. 3 thatincreased oxygen saturation and increased expired NO.

DETAILED DESCRIPTION OF THE INVENTION

[0013] As used herein, the term “treating” includes alleviating thesymptoms associated with a specific disorder or condition and/orpreventing or eliminating said symptoms. In particular, treating CFincludes causing one or more of the following: enhanced CFTR activity,augmented airway hydration, improved mucociliary clearance,bronchodilatation, and antimicrobial effect.

[0014] As used herein, an “effective amount” means an amount sufficientto produce a selected effect. For example, an effective amount of GSNOis an amount sufficient to alleviate the symptoms associated with CF.

[0015] The present invention is directed to use of a compositioncomprising a S-nitrosothiol in a form suitable for administration to aCF patient and formulated to maize contact with epithelial surfaces ofthe respiratory tract. S-Nitrosoglutathione is the most abundant ofseveral endogenous S-nitrosothiols. It is uniquely stable compared, forexample, to S-nitrosocysteine unless specific GSNO catabolic enzymes areupregulated. Such enzymes can include γ-glutamyl-transpeptidase,glutathione-dependent formaldehyde dehydrogenase, andthioredoxin-thioredoxin reductase. Additionally, there is evidence thatprokaryotic enzymes may exist which may limit the bactericidal effect ofGSNO. For this reason, coadministration of inhibitors of GSNOprokaryotic or eukaryotic GSNO catabolism may at times be necessary.This kind of inhibitor would include, but not be limited to, acivicingiven as 0.05 ml/kg of a 1 mM solution to achieve an airwayconcentration of 1 μM.

[0016] In part, the administration of a S-nitrosothiol reverses-the ionchannel abnormality associated with CF and alleviates thebronchoconstriction characteristic of CF. The present methodologyprovides a new route of antimicrobial therapy for CF, of particularrelevance because many patients have organisms that are resistant to allavailable conventional antimicrobial agents. The present invention alsodelivers nitrogen oxide to the CF airway, and surprisingly, increasesthe expression and maturation of CFTR.

[0017] Additional NO donors and transnitrosating agents suitable for usein the present invention in place of GSNO include compounds of thegeneral formula X—NO, where X=RS, and R is selected from the groupconsisting of cysteine, homocysteine, N-acetyl cysteine, cysteinylglycine, albumin, and other proteins; alternatively, X is selected fromthe group consisting of ethyl, propyl, tertbutyl, methyl, and otheralkyl hydrocarbons. Also included as an active compound isnitroglycerine, because both the nitro moieties (as nitrosating species)and the glycerin (metabolized to glycerol) can increase the expressionof CFTR.

[0018] In one embodiment, the invention consists of the delivery ofS-nitrosoglutathione in concentrations equal to or in excess of 500nmole/kg (175 mcg/kg), or other nitrosylating agents such as ethylnitrate or ethyl nitrite, to epithelial surfaces of patients with cysticfibrosis. The method provides adequate concentrations of S-nitrosylatingagent on the epithelial surfaces of CFTR and epithelial function toalleviate CF symptoms. These nebulized treatments, including GSNO, otherSNO's, and/or other NO⁺ donors with or without an inhibitor of breakdowncan be given on a periodic basis such as every 4, 6, or 8 hours toprevent accumulation of mucus, bronchoconstriction and bacterial growthin the CF airway, or given acutely during an exacerbation ofbronchoconstriction, mucus plugging, and bacterialbronchitis/bronchiolitis to patients with cystic fibrosis. Accordingly,the S-nitrosylating agents of the present invention can be administeredin an aerosolized form as preventive therapy for cystic fibrosis lungdisease, or as acute therapy for cystic fibrosis lung disease or toprovide relief from chronic obstructive pulmonary disease andbronchiectasis.

[0019] In accordance with one embodiment aerosolizedS-nitrosoglutathione and/or other S-nitrosothiols are administered in aconcentration of 10 mM using a dose of 0.05 ml/kg according to thefollowing calculation:

[0020] a. S-Nitrosoglutathione levels in the airways of normal subjectsare generally between 200 nM and 2 μM (in specimens undiluted bybronchoalveolar ravage).

[0021] b. Airway S-nitrosoglutathione levels in patients with pneumoniamay be in excess of 10 μM.

[0022] c. Airway lining fluid/extra cellular lung water volume is on theorder of 10 ml/kg in normal subjects.

[0023] d. Maximal direct delivery to the lower airway using conventionalnebulizer systems is 20% of the administered dose.

[0024] e. S-Nitrosoglutathione has a IC₅₀ in relaxing human airwaysmooth muscle preparations of approximately 10 μM.

[0025] f. Therefore, providing (5×10⁻⁵ l/kg×1×10⁻² M GSNO in 1×10⁻² l/kgvolume)×0.2=10×10⁻⁶ M concentration in the airway, in thephysiologically relevant and pharmacologically active range.

[0026] In one preferred embodiment an aerosol of 10 mM GSNO, at a doseof 0.05 ml/kg, is delivered to the CF airway on a daily basis.Additional embodiments include 1) the use of a similar concentration bydry powdered inhaler, and 2) delivery of greater concentrations to thelower respiratory tract by an aerosol, bronchoscopy, 3) endoscopicretrograde cholangiopancreatographic delivery, 4) delivery of an aerosolpreparation through the gastrointestinal mucosa, 5) by atomizer to thenasal mucosa and osteomeatal complex, 6) to the eustachian tube, to thevas deferens, cervix, or oviduct. This dosing has as its objectiverestoring normal levels of GSNO to the airway. In this regard, we haveshown that the majority of patients (15 out of 17) with mild cysticfibrosis undergoing bronchoalveolar lavage have undetectable SNO levelsin their airways whereas the majority of controls have detectable levels(as high as 1 μM, despite significant dilution effects involved in thetechnique) (FIG. 2.)

[0027] A broad range of symptoms associated with CF can treated by useof an inhaled nitrosylating agent. For example, the use of inhaled GSNOin doses of about 0.15 to about 0.03, more preferably about 0.05 ml/kgof a 10 mM concentration can be used to treat bronchoconstriction(airway smooth muscle tightening), and to treat hypoxia in cysticfibrosis. In addition, inhaled S-nitrosoglutathione can be used toprevent the long term deterioration of lung function, dyspnea, cough,chronic airway infection, bronchiectasis, atelectasis, pneumothorax, andrespiratory failure in patients with cystic fibrosis. These conditionsarise because of a defect in maturation and expression of functionalCFTR in the airway epithelium The ideal dose is 0.05 ml/kg of 10 mM GSNOdelivered by nebulization. Alternative doses may be applied in certaincircumstances.

[0028] In accordance with one embodiment, the S-nitrosothiolcompositions used in the present invention are formulated in an oraldosage form. Of note, GSNO is stable at acid pH. Most patients withcystic fibrosis have decreased pancreatic bicarbonate secretion and GSNOis anticipated to be bioavailable in the digestive system past the levelof the duodenum. The ideal dose is on the order of 150 mcg/kg. This canbe given up to 6 times a day. However, different doses and frequenciesmay at times be required and/or also claimed. The method comprisesadministering GSNO in an oral dosage from for increasinggastrointestinal epithelial function in cystic fibrosis patients withmeconium ileus, meconium ileus equivalent, and/or refractorymalabsorption. In another embodiment, surgical installation ofS-nitrosothiols, such as S-nitrosoglutathione, in patients with cysticfibrosis can be conducted using dysfunctional tubular epithelialstructures, including endoscopic injection in the drainage system of theparanatal sinuses, in the sinuses themselves, in the eustachian tubes,in the hepatobiliary collecting system, in the pancreatic dust, in theuterine cervix, in the fallopian tubes. In addition, surgicalinstallation into the vas deferens for the purpose of increasing thefunction of epithelium rendered incapable of hydrating secretions andclearing viscus secretions by virtue of mutations associated with lossof function of the CFTR protein is also encompassed in the presentinvention. For example, aqueous S-nitrosothiol preparations can be usedin surgical processes for cleaning sinuses and sinus osteae andeustachian tubes, for endoscopic retrograde cholangiopancreatography(ERCP) treatment of cholestasia arising from hepatobiliary sludging, andfor treatment and prevention of pancreatitis, for endoscopic treatmentof inspissates mucus in the uterine cervix or oviduct and for treatmentand prevention or infertility in men arising from inspecific mucus inthe vas deferens—all these indications are limited to patients having CFcharacterized by abnormal CFTR expression.

[0029] Many variations will be obvious to those skilled in the art.Therefore, the invention is defined by the claims.

1. A method for treating cystic fibrosis, said method comprising thestep of administering a composition comprising a nitrosylating agent toa patient having cystic fibrosis.
 2. The method according to claim 1wherein the nitrosylating agent is selected from the group consisting ofS-nitrosoglutathione and ethyl nitrite.
 3. The method according to claim2 wherein the composition is formulated as a powder or an aerosol andadministered to the nasal mucosa and osteomeatal complex.
 4. The methodaccording to claim 1 wherein the composition comprises about 100 toabout 150 mcg/kg of GSNO and the composition is delivered by drypowdered inhaler, metered dose inhaler, or alternative nebulizationsystems selected from the group consisting of ultrasonic, supersonic, orbreath actuated nebulizer.
 5. The method of claim 1 wherein thecomposition further comprises inhibitors of enzymes that catabolizeS-nitrosothiols.
 6. The method of claim 5 wherein the compositioncomprises 0.05 ml/kg of 10 mM GSNO in conjunction with bathocouproinedisulfinate and/or aurothioglucose.
 7. A method of enhancing theexpression and maturation of CFTR, said method comprising contacting therespiratory epithelium with a composition comprising a nitrosylatingagent.
 8. The method according to claim 7 wherein the nitrosylatingagent is selected from the group consisting of S-nitrosoglutathione andethyl nitrite.